Bacterial diversity within the human subgingival crevice

Abstract

Molecular, sequence-based environmental surveys of microorganisms have revealed a large degree of previously uncharacterized diversity. However, nearly all studies of the human endogenous bacterial flora have relied on cultivation and biochemical characterization of the resident organisms. We used molecular methods to characterize the breadth of bacterial diversity within the human subgingival crevice by comparing 264 small subunit rDNA sequences from 21 clone libraries created with products amplified directly from subgingival plaque, with sequences obtained from bacteria that were cultivated from the same specimen, as well as with sequences available in public databases. The majority (52.5%) of the directly amplified 16S rRNA sequences were <99% identical to sequences within public databases. In contrast, only 21.4% of the sequences recovered from cultivated bacteria showed this degree of variability. The 16S rDNA sequences recovered by direct amplification were also more deeply divergent; 13.5% of the amplified sequences were more than 5% nonidentical to any known sequence, a level of dissimilarity that is often found between members of different genera. None of the cultivated sequences exhibited this degree of sequence dissimilarity. Finally, direct amplification of 16S rDNA yielded a more diverse view of the subgingival bacterial flora than did cultivation. Our data suggest that a significant proportion of the resident human bacterial flora remain poorly characterized, even within this well studied and familiar microbial environment.

Figure 1

Streptococcus spp. (yellow) in a thin smear of the gingival specimen revealed by in situ hybridization with a Streptococcus-specific Texas Red-labeled oligonucleotide probe (red) and a broad range bacterial FITC-labeled oligonucleotide probe (green). (×500.)

Figure 2

Phylogenetic relationships of bacteria detected in a subgingival specimen, inferred from 16S rDNA sequence analysis. This unrooted tree was constructed by using 489 homologous sequence positions (534–1,050; E. coli numbering) and a maximum-likelihood algorithm. Sequences were given a colored alphanumeric designation according to whether they were amplified directly from the specimen (A; red), obtained from organisms cultivated from the specimen (C; green), or found by both methods (AC; blue). An organism name is provided adjacent to an alphanumeric designation when a sequence generated from this study was ≥99.0% identical to a sequence in a public database. Sequences selected from a public database for purposes of reference (not detected in the specimen) are indicated with the organism name in black. The number within each shaded fan indicates the total number of unique (amplified and cultivated) phylotypes within the group.

Figure 3

Phylogenetic relationships of bacteria that belong within the Low GC Gram Positive division, inferred from 16S rDNA sequence analysis. This tree was constructed by using 860 homologous sequence positions (534–1,400; E. coli numbering) and a maximum-likelihood algorithm; it was rooted by using the sequence of Peptococcus niger. Confidence values >50% from bootstrapped data are displayed. For sequence designation scheme, see Fig. 2 legend. Cluster designations are based on the proposed phylogenetic scheme of Collins et al. (26).

Figure 4

Phylogenetic relationships of bacteria that belong within the Actinobacteria division, inferred from 16S rDNA sequence analysis. This tree was constructed by using 894 homologous positions (534–1,438; E. coli numbering) and a maximum-likelihood algorithm; it was rooted by using the sequence of Streptomyces coelicolor. Confidence values >50% from bootstrapped data are displayed. For sequence designation scheme, see Fig. 2 legend.